Metabolic labeling of hyaluronan: Biosynthesis and quantitative analysis of 13C,15N-enriched hyaluronan by NMR and MS-based methods.

Hyaluronan (HA), a member of the GAG family of glycans, has many diverse biological functions that vary a lot depending on the length of the HA chain and its concentration. A better understanding of the structure of different-sized HA at the atomic level is therefore crucial to decipher these biological functions. NMR is a method of choice for conformational studies of biomolecules, but there are limitations due to the low natural abundance of the NMR active nuclei 13C and 15N. We describe here the metabolic labeling of HA using the bacterium Streptococcus equi subsp. Zooepidemicus and the subsequent analysis by NMR and mass spectrometry. The level of 13C and 15N isotope enrichment at each position was determined quantitatively by NMR spectroscopy and was further confirmed by high-resolution mass spectrometry analysis. This study provides a valid methodological approach that can be applied to the quantitative assessment of isotopically labeled glycans and will help improve detection capabilities and facilitate future structure-function relationship analysis of complex glycans.

Structure and Immunogenicity of the Bordetella pertussis LOS-Derived Oligosaccharides in the Endosomal-Like Pre-Processing Mice Model.

Glycoproteins are processed endosomally prior to presentation to T cells and subsequent induction of specific antibodies. The sugar part of glycoconjugate may be degraded while the type of the process depends on the features of the particular structure. The generated carbohydrate epitopes may differ from native structures and influence immunogenicity of the antigens. We have devised a model of endosomal-like pre-processing of Bordetella pertussis 186 oligosaccharides (OSs) to verify how it affects the immunogenicity of their conjugates. The glycoconjugates of structurally defined forms of the dodecasaccharide OS were synthesized and their immunogenicity was assessed using immunochemical methods. The structural features of the oligosaccharides and their sensitivity to deamination were analyzed by NMR spectroscopy. The distal trisaccharide-comprising pentasaccharide conjugated to a protein was the most effective in inducing immune response against the B. pertussis 186 LOS and the immune response to the complete OS conjugates was significantly lower. This could be explained by the loss of the distal trisaccharide during the in-cell deamination process suggesting that the native structure is not optimal for a vaccine antigen. Consequently, our research has shown that designing of new glycoconjugate vaccines requires the antigen structures to be verified in context of possible endosomal reactions beforehand.

Conserved Structural Features of Core Oligosaccharides among the Lipopolysaccharides of Respiratory Pathogens from the Genus Bordetella Analyzed Exclusively by NMR Spectroscopy.

Bacterial pathogens expose on the cell surface a variety of complex carbohydrate molecules. Gram-negative bacteria produce lipopolysaccharides, which are the main components of the outer membrane of bacterial envelopes and play a major role in host-pathogen interactions. B. pertussis, B. parapertussis, B. bronchiseptica, and B. holmesii, are mammalian respiratory pathogens, having substantial economic impact on human health and agriculture. B. pertussis is responsible for whooping cough (pertussis) and B. holmesii is the second pertussis etiological factor, but the current anti-pertussis vaccines do not provide cross-protection. The structural data on any given hypothetical carbohydrate antigen is a prerequisite for further analysis of structure-related activities and their interaction with hosts. 1H NMR spectra constitute fingerprints of the analyzed glycans and provide unique identity information. The concept of structure-reporter groups has now been augmented by 1H,13C-correlation spectra of the Bordetella oligosaccharides. The comparative analysis of Bordetellae oligosaccharides (OS) revealed that the hexasaccharide, comprising the α-GlcpN, α-GlcpA, 4,6-disubstituted-ß-Glcp, 2,7-disubstituted-l-α-d-Hepp, 3,4-disubstituted-l-α-d-Hepp, and Kdo, constitute the least variable OS segment. This minimal common element in the structure of lipopolysaccharides of Bordetellae could be used to devise a universal cross-protective vaccine component against infections with various bacteria from the genus Bordetella.

Bordetella holmesii Lipopolysaccharide Hide and Seek Game with Pertussis: Structural Analysis of the O-Specific Polysaccharide and the Core Oligosaccharide of the Type Strain ATCC 51541.

Whooping cough is a highly contagious disease caused predominantly by Bordetella pertussis, but it also comprises of a pertussis-like illness caused by B. holmesii. The virulence factors of B. holmesii and their role in the pathogenesis remain unknown. Lipopolysaccharide is the main surface antigen of all Bordetellae. Data on the structural features of the lipopolysaccharide (LPS) of B. holmesii are scarce. The poly- and oligosaccharide components released by mild acidic hydrolysis of the LPS were separated and investigated by 1H and 13C NMR spectroscopy, mass spectrometry, and chemical methods. The structures of the O-specific polysaccharide and the core oligosaccharide of B. holmesii ATCC 51541 have been identified for the first time. The novel pentasaccharide repeating unit of the B. holmesii O-specific polysaccharide has the following structure: {→2)-α-l-Rhap-(1→6)-α-d-Glcp-(1→4)-[ß-d-GlcpNAc-(1→3]-α-d-Galp-(1→3)-α-d-GlcpNAc-(1→}n. The SDS-PAGE and serological cross-reactivities of the B. holmesii LPS suggested the similarity between the core oligosaccharides of B. holmesii ATCC 51541 and B. pertussis strain 606. The main oligosaccharide fraction contained a nonasaccharide. The comparative analysis of the NMR spectra of B. holmesii core oligosaccharide fraction with this of the B. pertussis strain 606 indicated that the investigated core oligosaccharides were identical.

De­O­acylated lipooligosaccharide of E. coli B reduces the number of metastatic foci via downregulation of myeloid cell activity.

Lipopolysaccharides are the main surface antigens and virulence factors of gram­negative bacteria. Removal of four ester­bound fatty acid residues from hexaacyl lipid A of Escherichia coli lipooligosaccharide (LOS) resulted in the de­O­acylated derivative E. coli LOS­OH (LOS­OH). This procedure caused a significant reduction in the toxicity of this compound compared to the native molecule. We investigated the effect of such a structural LOS modification on its biological activity using in vitro assays with monocytic cells of the RAW264.7 line, dendritic cells of the JAWS II line, bone marrow­derived dendritic cells (BM­DCs), and spleen cells. Furthermore, in in vivo experiments with a melanoma B16 metastasis model, the anti­metastatic activity of the compounds and spleen cell reactivity mediated by them representing a systemic response were analyzed. The results revealed that LOS­OH demonstrated weaker ability than LOS to stimulate and polarize an immune response both in vitro and in vivo. It induced lower cytokine production by cells of myeloid lines. Multiple applications of LOS­OH into mice injected intravenously with B16 cells significantly (P<0.05; P<0.01) reduced the number of metastatic foci in the lungs, presumably via silencing of myeloid cell reactivity as well as the inability to stimulate lymphoid cells both directly and indirectly. These findings suggest that LOS­OH maintained in the body of metastasis­bearing mice appears to modulate or downregulate the innate response, leading to the inability of blood myeloid cells to support the migration of melanoma cells to lung tissue.

Structural Masquerade of Plesiomonas shigelloides Strain CNCTC 78/89 O-Antigen-High-Resolution Magic Angle Spinning NMR Reveals the Modified d-galactan I of Klebsiella pneumoniae.

The high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) analysis of Plesiomonas shigelloides 78/89 lipopolysaccharide directly on bacteria revealed the characteristic structural features of the O-acetylated polysaccharide in the NMR spectra. The O-antigen profiles were unique, yet the pattern of signals in the, spectra along with their ¹H,13C chemical shift values, resembled these of d-galactan I of Klebsiella pneumoniae. The isolated O-specific polysaccharide (O-PS) of P. shigelloides strain CNCTC 78/89 was investigated by ¹H and 13C NMR spectroscopy, mass spectrometry and chemical methods. The analyses demonstrated that the P. shigelloides 78/89 O-PS is composed of →3)-α-d-Galp-(1→3)-ß-d-Galf2OAc-(1→ disaccharide repeating units. The O-acetylation was incomplete and resulted in a microheterogeneity of the O-antigen. This O-acetylation generates additional antigenic determinants within the O-antigen, forms a new chemotype, and contributes to the epitopes recognized by the O-serotype specific antibodies. The serological cross-reactivities further confirmed the inter-specific structural similarity of these O-antigens.

Identification of d-Galactan-III As Part of the Lipopolysaccharide of Klebsiella pneumoniae Serotype O1.

Klebsiella pneumoniae is a Gram-negative, ubiquitous bacterium capable of causing severe nosocomial infections in individuals with impaired immune system. Emerging multi-drug resistant strains of this species and particularly carbapenem-resistant strains pose an urgent threat to public health. The lipopolysaccharide (LPS) O-antigen is the main surface antigen. It contributes to the virulence of this species and determines the O-serotype of K. pneumoniae isolates. Among the nine main O-serotypes of K. pneumoniae, O1-and O2-type pathogens are causative agents of over 50% of all infections. Serotype O1, the most common O-serotype, expresses complex LPS consisting of d-galactan-I (a polymer built of → 3)-ß-d-Galf-(1 → 3)-α-d-Galp-(1 → repeating units) capped by d-galactan-II (built of [ → 3)-α-d-Galp-(1 → 3)-ß-d-Galp-(1 →] repeating units). Galactan-I is present as the sole polymer in O2 serotype. Recently, in case of serotype O2, conversion of galactan-I to galactan-III (→ 3)-ß-d-Galf-(1 → 3)-[α-d-Galp-(1 → 4)]-α-d-Galp-(1 →) was reported. Substitution of → 3)-α-d-Galp by a branching terminal α-d-Galp was dependent on the presence of the gmlABC operon and had a major impact on the antigenicity of the galactan polymer. Genetic analysis indicated that 40% of the O1 clinical isolates also carry the gmlABC locus; therefore we aimed to characterize the corresponding phenotype of LPS O-antigens. The presence of galactan-III among O1 strains was proven using galactan-III-specific monoclonal antibodies and confirmed by structural analyses performed using sugar and methylation analysis as well as classical and high-resolution magic angle spinning NMR spectroscopy. By using an isogenic mutant pair, we demonstrated that galactan-III expression was dependent on the presence of glycosyltransferases encoded by gmlABC, as was shown previously for the O2 serotype. Furthermore, the galactan-II structures in O1gml+ strains remained unaffected corroborating no functional interactions between the biosynthesis of galactan-III and galactan-II polymers.

The O-antigen of Plesiomonas shigelloides serotype O36 containing pseudaminic acid.

The structure of the repeating unit of O-antigen of Plesiomonas shigelloides serotype O36 has been investigated by 1H and 13C NMR spectroscopy, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry and chemical methods. The new structure of trisaccharide has been established: [Formula: see text] These trisaccharide O-antigen units substitute the core undecasaccharide at C-4 of the ß-D-GlcpNAc residue. The core oligosaccharide and lipid A are identical with these of the serotype O17 (PCM 2231) (Maciejewska, A., Lukasiewicz, J., Kaszowska, M., Jachymek, W., Man-Kupisinska, A.; Lugowski, C. Mar. Drugs.2013, 11 (2), 440-454; Lukasiewicz, J., Dzieciatkowska, M., Niedziela, T., Jachymek, W., Augustyniuk, A., Kenne, L., Lugowski, C. Biochemistry, 2006, 45, 10434-10447).

Genetic Diversity of O-Antigens in Hafnia alvei and the Development of a Suspension Array for Serotype Detection.

Hafnia alvei is a facultative and rod-shaped gram-negative bacterium that belongs to the Enterobacteriaceae family. Although it has been more than 50 years since the genus was identified, very little is known about variations among Hafnia species. Diversity in O-antigens (O-polysaccharide, OPS) is thought to be a major factor in bacterial adaptation to different hosts and situations and variability in the environment. Antigenic variation is also an important factor in pathogenicity that has been used to define clones within a number of species. The genes that are required to synthesize OPS are always clustered within the bacterial chromosome. A serotyping scheme including 39 O-serotypes has been proposed for H. alvei, but it has not been correlated with known OPS structures, and no previous report has described the genetic features of OPS. In this study, we obtained the genome sequences of 21 H. alvei strains (as defined by previous immunochemical studies) with different lipopolysaccharides. This is the first study to show that the O-antigen gene cluster in H. alvei is located between mpo and gnd in the chromosome. All 21 of the OPS gene clusters contain both the wzx gene and the wzy gene and display a large number of polymorphisms. We developed an O serotype-specific wzy-based suspension array to detect all 21 of the distinct OPS forms we identified in H. alvei. To the best of our knowledge, this is the first report to identify the genetic features of H. alvei antigenic variation and to develop a molecular technique to identify and classify different serotypes.

[Bordetella pertussis lipooligosaccharide-derived neoglycoconjugates - new components of pertussis vaccine]. / Neoglikokoniugaty lipooligosacharydu Bordetella pertussis-nowe potencjalne skladniki szczepionki przeciwkrztuscowej.

Pertussis is a contagious respiratory tract disease caused by the Gram-negative bacterium Bordetella pertussis. Despite widespread vaccination, in recent years the pertussis incidence has increased. The whole-cell pertussis vaccine has been very effective but reactogenic. Therefore the improved vaccines contain only a few isolated and inactivated antigens of B. pertussis. However, a waning of the acellular vaccine-induced immunity indicates that these vaccines lack some important protective B. pertussis antigens. The vaccine containing an inactivated pertussis toxin induces the production of toxin-neutralizing antibodies, but it does not lead to destruction of bacteria. Since many virulence factors are involved in the pathogenesis of pertussis, beside the toxin-neutralizing activity, the direct bactericidal activity is essential in anti-pertussis immunity. Lipooligosaccharide is the main surface component of B. pertussis. It is a target for bactericidal antibodies during natural infection. The endotoxic activity of LOS makes it unacceptable for acellular vaccines against B. pertussis. However, the non-toxic moiety of the B. pertussis LOS-derived oligosaccharide coupled to a carrier protein forms an immunogenic glycoconjugate which has a potential application as a new component of a pertussis vaccine. In this paper, we present a review of current research and reasons for the increased pertussis incidence. The epidemiologic situation of pertussis in the past decades showing the ineffectiveness of contemporary, acellular pertussis vaccines is also discussed. The immune processes elicited by natural infection with B. pertussis were compared to the vaccine-induced immunity. The important role of bactericidal antibodies against lipooligosaccharide was indicated in effective immune defense. In a number of research papers the immunogenicity and protective properties of glycoconjugates containing the oligosaccharide component of B. pertussis have been described, and its application as a new component of a pertussis vaccine have been implied.

Structural studies of Helix aspersa agglutinin complexed with GalNAc: A lectin that serves as a diagnostic tool.

Lectins belong to a differentiated group of proteins known to possess sugar-binding properties. Due to this fact, they are interesting research targets in medical diagnostics. Helix aspersa agglutinin (HAA) is a lectin that recognizes the epitopes containing α-d-N-acetylgalactosamine (GalNAc), which is present at the surface of metastatic cancer cells. Although several reports have already described the use of HAA as a diagnostic tool, this protein was not characterized on the molecular level. Here, we present for the first time the structural information about lectin isolated from mucus of Helix aspersa (garden snail). The amino acid sequence of this agglutinin was determined by Edman degradation and tertiary as well as quaternary structure by X-ray crystallography. The high resolution crystal structure (1.38Å) and MALDI-TOF mass spectrometry analysis provide the detailed information about a large part of the HAA natural glycan chain. The topology of the GalNAc binding cleft and interaction with lectin are very well defined in the structure and fully confirmed by STD HSQC NMR spectroscopy. Together, this provides structural clues regarding HAA specificity and opens possibilities to rational modifications of this important diagnostic tool.

Core oligosaccharide of Escherichia coli B-the structure required for bacteriophage T4 recognition.

The structure of Escherichia coli B strain PCM 1935 core oligosaccharide has been investigated by (1)H and (13)C NMR spectroscopy, MALDI-TOF MS and ESI MS(n). It was concluded that the core oligosaccharide is a pentasaccharide with the following structure: ESI MS/MS analysis revealed that the glycine (a minor component) is linked to the →3,7)-l-α-d-Hepp-(1→ residue.

Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.

Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

The novel structure of the core oligosaccharide backbone of the lipopolysaccharide from the Plesiomonas shigelloides strain CNCTC 80/89 (serotype O13).

The new structure of the core oligosaccharide of Plesiomonas shigelloides CNCTC 80/89 (serotype O13) lipopolysaccharide has been investigated by chemical methods, (1)H and (13)C NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight (MALDI-TOF). It was concluded that the core oligosaccharide of P. shigelloides CNCTC 80/89 is a nonasaccharide with the following structure: The position of glycine was determined by MALDI-TOF MS/MS analyses.

The unique structure of complete lipopolysaccharide isolated from semi-rough Plesiomonas shigelloides O37 (strain CNCTC 39/89) containing (2S)-O-(4-oxopentanoic acid)-α-D-Glcp (α-D-Lenose).

The complete structure of semi-rough lipopolysaccharide (SR-LPS) of Plesiomonas shigelloides CNCTC 39/89 (serotype O37) has been investigated by (1)H and (13)C NMR spectroscopy, matrix-assisted laser-desorption/ionization time-of-flight MS, and chemical methods. The following structure of the single unit of the O-antigen has been established: [formula see text] in which α-D-Lenp is (2S)-O-(4-oxopentanoic acid)-α-D-Glcp residue which has not been found in nature. The absolute configuration of oxopentanoic acid moiety in α-d-Lenose residue was determined by NOESY experiment combined with molecular modeling (MM2 force field). The decasaccharide core is substituted at C-4 of the ß-D-Glcp residue with a single pentasaccharide unit. Lipid A is built of a ß-D-GlcpN4P-(1→6)-α-D-GlcpN1P disaccharide asymmetrically substituted with fatty acids. It was concluded that the core oligosaccharide and the lipid A are identical with those in P. shigelloides CNCTC 113/92 Niedziela et al. (2002)(9) and Lukasiewicz et al. (2006).(10.)

New functional ligands for ficolin-3 among lipopolysaccharides of Hafnia alvei.

Ficolin-1 (M), ficolin-2 (L), ficolin-3 (H) and mannan-binding lectin (MBL) activate the complement system and have opsonic activity. The specificity of ficolin-3 is poorly characterized and currently limited to a few ligands only. We present new specific targets for human ficolin-3, identified among lipopolysaccharides (LPSs, endotoxin) of Hafnia alvei. The interaction was restricted to LPSs of four strains: 23, Polish Collection of Microorganisms (PCM) 1200, PCM 1203 and PCM 1205 and limited to their O-specific polysaccharides (O-specific PSs) composed of different numbers of oligosaccharide (OS) repeating units (RUs). Moreover, these LPS/ficolin-3 complexes activated the lectin pathway of complement in a C4b-deposition assay in a calcium- and magnesium-dependent way. A neoglycoconjugate of the O-specific PS fraction of H. alvei 1200 LPS with bovine serum albumin (BSA) was prepared and used as a tool for the determination of ficolin-3 concentration and activity in serum. To confirm a structure of the O-specific PS 1200 selected for the conjugate preparation, structural analysis was performed on a series of O-specific PSs released by the mild acid hydrolysis of the LPS. The isolated O-specific PSs, showing the different length distributions, were devoid of a major part of the core OS region and had Hep-Kdo disaccharide at a reducing end. The neoglycoconjugate was a highly selective tool for the determination of ficolin-3 concentration and activity in serum (lectin pathway activation in the C4b deposition assay) and was not affected by MBL, ficolin-1 and ficolin-2 or natural antibodies.

Elevated levels of anti-endotoxin antibodies in patients with bilateral idiopathic acute anterior uveitis.

PURPOSE: Endotoxins have been proved to be responsible for acute anterior uveitis (AAU) in animals in a well-established experimental model of endotoxin-induced uveitis (EIU). The purpose of our study was the detection of antibodies against endotoxins of selected enterobacteria in the serum of patients with idiopathic AAU and searching for correlations between the levels of these antibodies and the presence of HLA-B27 antigen as well as characteristic signs of EIU such as bilaterality and the absence of spontaneous recurrences of the disease. METHODS: Reactions of serum IgG antibodies with lipopolysaccharides (LPSs) of Escherichia coli O1, E. coli O10, E. coli O111, E. coli J5, and Klebsiella pneumoniae O3 were determined for 60 patients with idiopathic AAU and 40 healthy volunteers. The presence of HLA-B27 antigen in patients was determined. Documentation of the frequency of recurrences of AAU during a follow-up period of 8 years was collected. RESULTS: We have observed that the sera of patients with a first attack of AAU reacted stronger with the LPS of K. pneumoniae O3 than the sera of patients with relapse of the disease. Patients with bilateral AAU had markedly higher levels of antibodies against four of the five used LPSs than patients with one eye involved. A multiply comparison showed higher levels of IgG reacting with LPS of E. coli O111 in patients with bilateral eye inflammation admitted with the first attack of AAU comparing to controls. The incidence of recurrent form of AAU was significantly increased in HLA-B27-positive patients compared to HLA-B27-negative patients. However, we found in HLA-B27 carriers that those with the bilateral form of AAU had over three times smaller risk of recurrence and showed stronger immunization by endotoxins than patients with unilateral inflammation. CONCLUSION: Our results suggest a potential role of endotoxins in the aetiology of the nonrecurrent bilateral form of AAU. We suggest that not only HLA-B27 status but also determination of number of involved eyes may be useful to assess the risk of recurrence of the idiopathic AAU.

Structural analysis of the lipid A isolated from Hafnia alvei 32 and PCM 1192 lipopolysaccharides.

Hafnia alvei, a Gram-negative bacterium, is an opportunistic pathogen associated with mixed hospital infections, bacteremia, septicemia, and respiratory diseases. The majority of clinical symptoms of diseases caused by this bacterium have a lipopolysaccharide (LPS, endotoxin)-related origin. The lipid A structure affects the biological activity of endotoxins predominantly. Thus, the structure of H. alvei lipid A was analyzed for the first time. The major form, asymmetrically hexa-acylated lipid A built of beta-D-GlcpN4P-(1-->6)-alpha-D-GlcpN1P substituted with (R)-14:0(3-OH) at N-2 and O-3, 14:0(3-(R)-O-12:0) at N-2', and 14:0(3-(R)-O-14:0) at O-3', was identified by ESI-MS(n) and MALDI-time-of-flight (TOF) MS. Comparative analysis performed by MS suggested that LPSs of H. alvei 32, PCM 1192, PCM 1206, and PCM 1207 share the identified structure of lipid A. LPSs of H. alvei are yet another example of enterobacterial endotoxins having the Escherichia coli-type structure of lipid A. The presence of hepta-acylated forms of H. alvei lipid A resulted from the addition of palmitate (16:0) substituting 14:0(3-OH) at N-2 of the alpha-GlcpN residue. All the studied strains of H. alvei have an ability to modify their lipid A structure by palmitoylation.

Structures of two novel, serologically nonrelated core oligosaccharides of Yokenella regensburgei lipopolysaccharides differing only by a single hexose substitution.

Immunochemical analysis of the Yokenella regensburgei lipopolysaccharides (LPS) indicated the presence of the core oligosaccharide-related immunotypes among the investigated strains. The structure of the core oligosaccharide segment of the Y. regensburgei LPS has been investigated using chemical methods, mass spectrometry, and (1)H, (13)C NMR spectroscopy. It was concluded that the core oligosaccharides of the strains PCM 2476 and PCM 2477 are composed of an undecasaccharide. The combined data revealed two immunotypes of the core oligosaccharide recognized by antibodies against the whole bacterial cells. The structural differences between the core oligosaccharides are limited to the outermost terminal hexopyranose residue. In the core oligosaccharide of the strain PCM 2476, it was identified as alpha-d-Glcp and in that of the strain PCM 2477 as alpha-d-Galp. This subtle difference between the glycoforms of the LPS core appeared to be essential for formation of the epitopes recognized by the specific antibodies directed against the Y. regensburgei whole bacterial cells. The oligosaccharides are not substituted by phosphate groups. Instead, the carboxyl groups of Kdo and galacturonic acid residues present in the core provide the negative charges. The undecasaccharides represent a novel core type of bacterial LPS, which is characteristic for Y. regensburgei.